Multi-component hydrogel gels and uses thereof

Abstract

The present disclosure relates to multi-component hydrogels that can be used, for example, as dermal fillers for the treatment of fine and wrinkles of the skin. Also disclosed herein are methods for making and using the disclosed hydrogels and related compositions.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 408,718, filed on September 21, 2022, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to hydrogels and compositions comprising the hydrogels. The disclosed hydrogels and compositions can be used as dermal fillers or in cosmetic procedures, and they can be used to effectively treat fine lines and wrinkles on the skin. Background Art

[0004] The following discussion is provided only to assist the reader in understanding the present disclosure, and the following discussion does not admit that the description or constitutes prior art thereof.

[0005] Hydrophilic gels or hydrogels are widely used in the biomedical field, such as during adhesive surgery and as dermal fillers. They are typically prepared by chemical cross-linking of polymers. A variety of injectable dermal filler products have been developed for treating or correcting facial defects, such as wrinkles and volume loss due to the natural effects of aging. Injectable "dermal fillers" temporarily restore a smoother, more youthful appearance. Ideally, dermal fillers are long-lasting, soft, smooth, and natural when introduced into or under the skin.

[0006] Glycosaminoglycans (GAGs) or mucopolysaccharides are long linear polysaccharides composed of repeating disaccharide units (i.e., two sugar units). The repeating two-sugar units are composed of uronic acid and amino sugar. Hyaluronic acid (HA) (also known as hyaluronan) is a water-soluble glycosaminoglycan that is a major component of the extracellular matrix and is widely distributed in animal tissues. HA has excellent biocompatibility and does not cause allergic reactions when implanted into a patient. In addition, HA has the ability to bind a large amount of water, making it an excellent volumizer for soft tissues.

[0007] Alpha-hydroxy acids (AHAs) are a class of chemical compounds that consist of carboxylic acids with a hydroxyl group substituted on adjacent carbons. Prominent examples are glycolic acid, lactic acid, and citric acid. Polymers made from AHAs can serve as biocompatible materials. For example, poly-L-lactic acid (PLLA) is an immunologically inert, biocompatible, and biodegradable synthetic polymer. Once in the body, PLLA will eventually degrade and undergo absorption. Summary of the Invention

[0008] The present application provides hydrogels and compositions that include crosslinked glycosaminoglycans (e.g., hyaluronic acid or "HA"), uncrosslinked glycosaminoglycans (e.g., HA), and poly(α-hydroxy acid) polymers (e.g., poly-L-lactic acid or "PLLA"). The disclosed hydrogels have improved flowability and spreadability compared to products that include, for example, crosslinking between HA and PLLA. The disclosed hydrogels and compositions can be used as dermal fillers or skin boosters and are used to treat or reduce the appearance of fine lines (e.g., fine lines on the cheeks), acne scars, and skin quality. The disclosed hydrogels, as fillers, boost the quality of the skin and stimulate the skin for immediate improvement in skin texture and quality with long-lasting effects, and the disclosed hydrogels and compositions are particularly suitable for this use due to their spreadability, which provides an immediate lifting effect on the skin and continuously improves skin quality and long-term implant duration and stability / shelf life. The hydrogels and compositions disclosed in the present application improve skin quality attributes, including elasticity, glossiness (reducing dry skin and increasing hydration), texture, and smoothness (improving pore size, wrinkled and tissue-like skin), and skin color, for a duration greater than or equal to 9 months (e.g., up to 12 or 18 months).

[0009] The disclosed hydrogels and compositions are particularly suitable for this use due to their spreadability, which provides an immediate lifting effect on the skin and continuously improves skin quality and long-term implant duration and stability / shelf life. The hydrogels and compositions disclosed in the present application improve skin quality attributes, including elasticity, glossiness (reducing dry skin and increasing hydration), texture, and smoothness (improving pore size, wrinkled and tissue-like skin), and skin color, for a duration between greater than or equal to 9 months, such as 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or up to 24 months.

[0010] In one aspect, the present disclosure provides a composition that includes: (a) a crosslinked glycosaminoglycan hydrogel at 10 mg / ml to 45 mg / ml, (b) 20% to 50% by weight of the crosslinked glycosaminoglycan hydrogel of free, uncrosslinked glycosaminoglycan, and (c) a biodegradable poly(α-hydroxy acid) polymer at 10 mg / ml to 50 mg / ml, wherein the biodegradable poly(α-hydroxy acid) polymer does not crosslink with the glycosaminoglycan hydrogel or the free, uncrosslinked glycosaminoglycan.

[0011] Another aspect of the disclosure provides a composition prepared by the following method, the method comprising: (a) crosslinking a glycosaminoglycan in the presence of 2% to 4% v / v NaOH to obtain a crosslinked glycosaminoglycan hydrogel; (b) filtering the crosslinked glycosaminoglycan hydrogel using a 60 to 90 micron filter; (c) adding 20% to 50% by weight of the composition of free, non-crosslinked glycosaminoglycan to the filtered crosslinked glycosaminoglycan hydrogel; and (d) adding 10 mg / ml to 50 mg / ml of a biodegradable poly(α-hydroxy acid) polymer to the composition from (c) under vacuum conditions, wherein the biodegradable poly(α-hydroxy acid) polymer does not crosslink with the glycosaminoglycan hydrogel. In some embodiments, the method further comprises swelling the crosslinked glycosaminoglycan hydrogel under vacuum conditions between (a) and (b). In some embodiments, the method further comprises precipitating and washing the filtered crosslinked glycosaminoglycan hydrogel from (b) with ethanol. In some embodiments, the method further comprises sterilizing the composition after (d).

[0012] In some embodiments, the composition comprises 10 mg / ml to 20 mg / ml, 10 mg / ml to 25 mg / ml, 10 mg / ml to 30 mg / ml, 10 mg / ml to 35 mg / ml, 10 mg / ml to 40 mg / ml, 10 mg / ml to 45 mg / ml, 15 mg / ml to 20 mg / ml, 15 mg / ml to 25 mg / ml, 15 mg / ml to 30 mg / ml, 15 mg / ml to 35 mg / ml, 15 mg / ml to 40 mg / ml or 15 mg / ml to 45 mg / mL of the crosslinked glycosaminoglycan hydrogel.

[0013] In some embodiments, the composition comprises 20% to 30%, 20% to 35%, 20% to 40% or 20% to 45% by weight of the crosslinked glycosaminoglycan hydrogel of free, non-crosslinked glycosaminoglycan.

[0014] In some embodiments, the composition comprises 10 mg / ml to 15 mg / ml, 10 mg / ml to 20 mg / ml, 10 mg / ml to 25 mg / ml, 10 mg / ml to 30 mg / ml, 10 mg / ml to 35 mg / ml, 10 mg / ml to 40 mg / mg or 10 mg / ml to 50 mg / ml of the biodegradable poly(α-hydroxy acid) polymer.

[0015] In some embodiments, the crosslinked glycosaminoglycan hydrogel comprises crosslinked hyaluronic acid (HA), crosslinked heparan sulfate (HS), crosslinked heparin (HEP), crosslinked chondroitin sulfate (CS), crosslinked dermatan sulfate (DS), or crosslinked keratan sulfate (KS), or a combination thereof.

[0016] In some embodiments, the free glycosaminoglycan comprises hyaluronic acid (HA), heparan sulfate (HS), heparin (HEP), chondroitin sulfate (CS), dermatan sulfate (DS), or keratan sulfate (KS), or a combination thereof.

[0017] In some embodiments, the biodegradable poly(α-hydroxy acid) polymer comprises poly-L-lactic acid (PLLA), poly-D-lactic acid (PLDA), poly(glycolic acid) (PLGA), or poly(diethylene citrate) (PDC), or a combination thereof.

[0018] In some embodiments, the crosslinked glycosaminoglycan hydrogel has a size of about 60 to 90 microns, and optionally, wherein the biodegradable poly(α-hydroxy acid) polymer is about 40 to 63 microns.

[0019] In some embodiments, the crosslinked glycosaminoglycan is crosslinked by one or more multifunctional crosslinking agents.

[0020] In some embodiments, the one or more multifunctional crosslinking agents are selected from the group consisting of divinyl sulfone, polyepoxides, and diepoxides.

[0021] In some embodiments, the one or more multifunctional crosslinking agents are selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane.

[0022] In some embodiments, the crosslinked glycosaminoglycan is crosslinked by (i) a non-carbohydrate-based dinucleophilic or polynucleophilic crosslinking agent or (ii) a carbohydrate-based dinucleophilic or polynucleophilic crosslinking agent.

[0023] In some embodiments, the non-carbohydrate-based dinucleophilic or polynucleophilic crosslinking agent is hexamethylenediamine (HMDA).

[0024] In some embodiments, the carbohydrate-based dinucleophilic or polynucleophilic crosslinking agent is diaminotrehalose (DATH).

[0025] In some embodiments, the composition is an injectable composition.

[0026] Another aspect of the disclosure relates to a method for improving the skin quality of a subject in need of such treatment, which comprises administering to the subject a composition of the present disclosure.

[0027] Another aspect of the disclosure relates to a method for preparing a composition, which comprises: (a) crosslinking a glycosaminoglycan in the presence of 2% to 4% v / v NaOH to obtain a crosslinked glycosaminoglycan hydrogel; (b) filtering the crosslinked glycosaminoglycan hydrogel using a 60 to 90 micron filter; (c) adding 20% to 50% free glycosaminoglycan by weight of the composition to the filtered crosslinked glycosaminoglycan hydrogel; and (d) adding 10 mg / ml to 50 mg / ml of a biodegradable poly(α-hydroxy acid) polymer to the composition in step (c) under vacuum conditions, wherein the biodegradable poly(α-hydroxy acid) polymer does not crosslink with the glycosaminoglycan hydrogel.

[0028] In some embodiments, the method further comprises swelling the crosslinked glycosaminoglycan hydrogel under vacuum conditions between (a) and (b).

[0029] In some embodiments, the method further comprises precipitating and washing the filtered crosslinked glycosaminoglycan hydrogel from (b) with ethanol.

[0030] In some embodiments, the method further comprises sterilizing the composition after (d).

[0031] In some embodiments, the composition comprises 10 mg / ml to 20 mg / ml, 10 mg / ml to 25 mg / ml, 10 mg / ml to 30 mg / ml, 10 mg / ml to 35 mg / ml, 10 mg / ml to 40 mg / ml, 10 mg / ml to 45 mg / ml, 15 mg / ml to 20 mg / ml, 15 mg / ml to 25 mg / ml, 15 mg / ml to 30 mg / ml, 15 mg / ml to 35 mg / ml, 15 mg / ml to 40 mg / ml or 15 mg / ml to 45 mg / mL of the crosslinked glycosaminoglycan hydrogel.

[0032] In some embodiments, the composition comprises 20% to 30%, 20% to 35%, 20% to 40% or 20% to 45% free glycosaminoglycan by weight of the crosslinked glycosaminoglycan hydrogel.

[0033] In some embodiments, the composition comprises a biodegradable poly(α-hydroxy acid) polymer at 10 mg / ml to 15 mg / ml, 10 mg / ml to 15 mg / ml, 10 mg / ml to 20 mg / ml, 10 mg / ml to 25 mg / ml, 10 mg / ml to 30 mg / ml, 10 mg / ml to 35 mg / ml, 10 mg / ml to 40 mg / mg, or 10 mg / ml to 45 mg / ml.

[0034] In some embodiments, the crosslinked glycosaminoglycan hydrogel comprises crosslinked hyaluronic acid (HA), crosslinked heparan sulfate (HS), crosslinked heparin (HEP), crosslinked chondroitin sulfate (CS), crosslinked dermatan sulfate (DS), or crosslinked keratan sulfate (KS), or a combination thereof.

[0035] In some embodiments, the free glycosaminoglycan comprises hyaluronic acid (HA), heparan sulfate (HS), heparin (HEP), chondroitin sulfate (CS), dermatan sulfate (DS), or keratan sulfate (KS), or a combination thereof.

[0036] In some embodiments, the biodegradable poly(α-hydroxy acid) polymer comprises poly-L-lactic acid (PLLA), poly-D-lactic acid (PLDA), polyglycolic acid (PLGA), or poly(diethylene citrate) (PDC), or a combination thereof.

[0037] In some embodiments, the crosslinked glycosaminoglycan hydrogel has a size of about 60 to 90 microns, and optionally, wherein the biodegradable poly(α-hydroxy acid) polymer is about 40 to 63 microns.

[0038] In some embodiments, the crosslinked glycosaminoglycan is crosslinked by one or more multifunctional crosslinking agents.

[0039] In some embodiments, the one or more multifunctional crosslinking agents are selected from the group consisting of divinyl sulfone, polyepoxides, and diepoxides.

[0040] In some embodiments, the one or more multifunctional crosslinking agents are selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane.

[0041] In some embodiments, the crosslinked glycosaminoglycan is crosslinked by (i) a non-carbohydrate-based dinucleophilic or polynucleophilic crosslinking agent or (ii) a carbohydrate-based dinucleophilic or polynucleophilic crosslinking agent.

[0042] In some embodiments, the non-carbohydrate-based dinucleophile or polynucleophile crosslinker is hexamethylenediamine (HMDA).

[0043] In some embodiments, the carbohydrate-based dinucleophile or polynucleophile crosslinker is diaminotrehalose (DATH).

[0044] In some embodiments, the method further comprises formulating the composition into an injectable composition.

[0045] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages, and novel features will be apparent to those skilled in the art from the following drawings description and the specific embodiments of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The figures (drawings) illustrate a general representation of an exemplary method of manufacturing the disclosed hydrogels. DETAILED DESCRIPTION

[0047] The hydrogels and compositions provided by the present disclosure include crosslinked glycosaminoglycans (e.g., hyaluronic acid or "HA"), uncrosslinked glycosaminoglycans (e.g., HA), and poly(α-hydroxy acid) polymers (e.g., poly-L-lactic acid or "PLLA"). The disclosed hydrogels have firmness, fluidity, and spreadability, making them ideal for cosmetic and dermatological applications, including but not limited to treating or reducing the appearance of fine lines and wrinkles. In addition, the disclosed hydrogels and related compositions can be stable for more than 18 months, and this shelf life provides more commercial appeal for the disclosed compositions, which can be shipped in ampoules, vials, or pre-filled syringes.

[0048] Generally, the disclosed hydrogels and compositions are designed to deliver a constant dose of PLLA (e.g., by injection) during administration, while other hydrogels cannot. In other words, the PPLA particles are evenly distributed throughout the ampoule / syringe / vial from the beginning, middle, and end. After administration to a subject, the disclosed hydrogels and compositions improve skin quality attributes for a duration of greater than or equal to 9 months, such as 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or up to 24 months, including improving elasticity, gloss (reducing dry skin and increasing hydration), texture, and smoothness (improving pore size, reducing wrinkled and papery skin), and skin tone.

[0049] The disclosed hydrogels are formed using the following method: Crosslinked GAG particles (e.g., crosslinked HA) within a desired size range are prepared to prevent aggregation of poly(α-hydroxy acid) polymers (e.g., PLLA), thereby providing a more uniformly dispersed hydrogel, and the components are mixed under vacuum conditions during a specific step. Further, the addition of non-crosslinked GAG (e.g., "free HA") prevents unwanted stickiness, which aids in the manufacture, transportation, and end use of the hydrogel and hydrogel-containing products. In summary, the disclosed method can produce unique and desirable hydrogels that are suitable for any number of cosmetic and dermatological indications.

[0050] Definitions

[0051] As used herein, the singular forms "a / an" and "the" denote both the singular and the plural unless clearly indicated to mean only the singular.

[0052] It should be understood that although not always explicitly stated, all numerical designations are preceded by the term "about". The term "about" means that the understood numerical value is not limited to the exact number recited herein and is intended to refer to numbers that are substantially around the recited number without departing from the scope of the invention. As used herein, "about" will be understood by one of ordinary skill in the art and will vary somewhat depending on the context in which it is used. If the use of the term is unclear to one of ordinary skill in the art, then "about" will mean up to plus or minus 15%, 10%, 5%, 1%, or 0.1% of this particular term, considering the context in which the term is used.

[0053] Also as used herein, "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0054] As used herein, the terms "administerer", "administer", or "administering" mean (1) to provide, give, administer, and / or prescribe, such as by a health professional, or his or her authorized agent, or under his direction; (2) to introduce, take, or consume, such as by a health professional or a subject. Administration can be unrestricted and can be administered by subcutaneous, intramuscular, subcutaneous, intradermal, or transdermal injection. Administration can be unilateral or bilateral depending on the needs of a given patient.

[0055] Hydrogels and Compositions

[0056] The disclosed aspects relate to a composition comprising: a crosslinked glycosaminoglycan hydrogel at 10 mg / ml to 45 mg / ml; 20% to 50% by weight of free glycosaminoglycan based on the crosslinked glycosaminoglycan hydrogel; and a biodegradable poly(α-hydroxy acid) polymer at 10 mg / ml to 50 mg / ml, wherein the biodegradable poly(α-hydroxy acid) polymer is not crosslinked to the glycosaminoglycan hydrogel (i.e., it can be a free biodegradable poly(α-hydroxy acid) polymer). In some embodiments, the crosslinked glycosaminoglycan hydrogel is a crosslinked hyaluronic acid (HA) hydrogel. In some embodiments, the free glycosaminoglycan is free hyaluronic acid (HA). In some embodiments, the biodegradable poly(α-hydroxy acid) polymer is poly-L-lactic acid (PLLA). Thus, in some embodiments, the disclosed composition can comprise a crosslinked HA hydrogel at 10 mg / ml to 45 mg / ml, 20% to 50% by weight of HA based on the crosslinked HA hydrogel, and PLLA at 10 mg / ml to 50 mg / ml, wherein the PLLA is not crosslinked to the HA hydrogel.

[0057] The disclosed aspects relate to a composition prepared by a method comprising: (a) crosslinking a glycosaminoglycan in the presence of 2% to 4% (e.g., 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9% or 4%) v / v sodium hydroxide (NaOH) to obtain a crosslinked glycosaminoglycan hydrogel; (b) filtering the crosslinked glycosaminoglycan hydrogel using a filter of 60 to 90 microns (e.g., a 60, 70, 80 or 90 micron filter); (c) adding 20% to 50% (e.g., 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%) free glycosaminoglycan based on the weight of the crosslinked glycosaminoglycan hydrogel; and (d) adding 5 mg / ml to 60 mg / ml (5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml, 45 mg / ml, 46 mg / ml, 47 mg / ml, 48 mg / ml, 49 mg / ml, 50 mg / ml, 51 mg / ml, 52 mg / ml, 53 mg / ml, 54 mg / ml, 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml or 60 mg / ml) of a biodegradable poly(α-hydroxy acid) polymer to the composition of step (c) under vacuum conditions, wherein the biodegradable poly(α-hydroxy acid) polymer does not crosslink with the glycosaminoglycan hydrogel. In some embodiments, the method further comprises swelling the crosslinked glycosaminoglycan hydrogel under vacuum conditions between step (a) and step (b).In some embodiments, the method further comprises using ethanol to precipitate and wash the filtered crosslinked glycosaminoglycan hydrogel from step (b). In some embodiments, the method further comprises sterilizing the composition after step (d). In some embodiments, the glycosaminoglycan is HA. In some embodiments, the biodegradable poly(α-hydroxy acid) polymer is PLLA.

[0058] In some embodiments, the crosslinking in step (a) is achieved in the presence of 2.3% to 3.1% (e.g., 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3% or 3.1%) v / v NaOH. In some embodiments, the crosslinking in step (a) is achieved in the presence of 2.3% to 2.5% (e.g., 2.3%, 2.4% or 2.5%) v / v NaOH. In some embodiments, the crosslinking in step (a) is achieved in the presence of 2.4% NaOH.

[0059] In some embodiments, the composition is used as a dermal filler. The disclosed compositions show improved flowability and spreadability when administered as a filler under the skin of a patient. When used as a dermal filler, the disclosed compositions also show a more immediate and desirable "lift" due to the increased firmness of the gel. Further, the disclosed hydrogels have a low extrusion force (e.g., a force of less than 12 Newtons (12 N), such as 11 N, 10 N, 9 N, 8 N, 7 N, 6 N or less), and cause minimal clogging when using a fine needle (e.g., 25 to 30 gauge, such as 25 gauge or 27 gauge). In addition, the disclosed hydrogels are ready-to-use, i.e., they do not require reconstitution prior to administration. The firmness (G') and flexibility (xStrain) of HA-based filler products can be tested using rheometry. The G' value reflects the gel strength (firmness) of the product. xStrain is an indicator of gel flexibility (how much strain the gel can withstand and still be reversible). In some embodiments, the disclosed hydrogels show a G' value between 20 Pa and 200 Pa (e.g., 20 Pa, 30 Pa, 40 Pa, 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa, 100 Pa, 110 Pa, 120 Pa, 130 Pa, 140 Pa, 150 Pa, 160 Pa, 170 Pa, 180 Pa, 190 Pa or 200 Pa). In some embodiments, the disclosed hydrogels show a G' value between 30 Pa and 100 Pa (e.g., 30 Pa, 40 Pa, 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa or 100 Pa).). In some embodiments, the disclosed hydrogels show a G' value between 40 Pa and 80 Pa (e.g., 40 Pa, 50 Pa, 60 Pa, 70 Pa or 80 Pa). In some embodiments, the disclosed hydrogels show an xStrain value between 250% and 500% (e.g., 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 450%, 460%, 470%, 480%, 490% or 500%). In some embodiments, the disclosed hydrogels show an xStrain value between 300% and 400% (e.g., 300%, 310%, 320%, 330%, 350%, 360%, 370%, 380%, 390% or 400%). In some embodiments, the disclosed hydrogels show an xStrain value between 310% and 380% (e.g., 310%, 320%, 330%, 350%, 360%, 370% or 380%).

[0060] As used herein, the term "vacuum condition" refers to a condition where the air pressure is less than or equal to 30 mmHg. In some embodiments, the vacuum condition is achieved by a vacuum homogenizer. In some embodiments, the vacuum condition further includes a temperature of 68°C - 72°C (e.g., 68°C, 69°C, 70°C, 71°C, or 72°C).

[0061] In some embodiments, the crosslinked glycosaminoglycan and the free glycosaminoglycan are composed of the same type of glycosaminoglycan. In some embodiments, the crosslinked glycosaminoglycan is composed of a first type of glycosaminoglycan, and the free glycosaminoglycan is composed of a second type of glycosaminoglycan, wherein the first type of glycosaminoglycan is different from the second type of glycosaminoglycan. In some embodiments, the first glycosaminoglycan and the second glycosaminoglycan are HA.

[0062] In some embodiments, the glycosaminoglycan is gamma-irradiated. In some embodiments, the glycosaminoglycan is not gamma-irradiated. In some embodiments, the glycosaminoglycan is heat sterilized.

[0063] In some embodiments, the composition comprises from 5 mg / ml to 20 mg / ml (e.g., 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml), from 5 mg / ml to 15 mg / ml (e.g., 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, or 15 mg / ml), from 10 mg / ml to 20 mg / ml (e.g., 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml), from 10 mg / ml to 25 mg / ml (e.g., 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, or 25 mg / ml), from 10 mg / ml to 30 mg / ml (e.g., 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, or 30 mg / ml), from 10 mg / ml to 35 mg / ml (e.g., 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, or 35 mg / ml), from 10 mg / ml to 40 mg / ml (e.g.,10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml or 40 mg / ml), 15 mg / ml to 17 mg / ml (e.g., 15 mg / ml, 16 mg / ml or 17 mg / ml), 15 mg / ml to 20 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml or 20 mg / ml), 15 mg / ml to 25 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml or 25 mg / ml), 15 mg / ml to 30 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml or 30 mg / ml), 15 mg / ml to 35 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml or 35 mg / ml) or 15 mg / ml to 40 mg / ml (e.g.,Crosslinked glycosaminoglycan (e.g., HA) hydrogels at 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, or 40 mg / ml).

[0064] In some embodiments, the glycosaminoglycan is crosslinked with one or more crosslinking (e.g., multifunctional crosslinking) agents. The one or more multifunctional crosslinking agents are independently selected from the group consisting of divinyl sulfone, polyepoxides, and diepoxides. In certain embodiments, the multifunctional crosslinking agent is independently selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. In some embodiments, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). Thus, in some embodiments, the crosslinking agent includes an ether bond. In some embodiments, the concentration of BDDE used for crosslinking ranges between 0.001 and 0.05 g (e.g., 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, or 0.05 g) of BDDE per gram of glycosaminoglycan.

[0065] Additionally or alternatively, the crosslinking agent can comprise or consist of: (i) a spacer group and (ii) a functional group of the crosslinking agent that reacts with a carboxylic acid group on the GAG to form a linking group. For example, the spacer group can include a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. Thus, crosslinking can be achieved using a non-carbohydrate-based di- or poly-nucleophilic crosslinking agent (e.g., hexamethylenediamine (HMDA)) or a carbohydrate-based di- or poly-nucleophilic crosslinking agent (e.g., diaminotrehalose (DATH)) together with the glycosaminoglycan.

[0066] In some embodiments, the composition comprises from 20% to 30% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%), from 20% to 35% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35%), from 20% to 40% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%) or from 20% to 45% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45%) free glycosaminoglycan, by weight of the crosslinked glycosaminoglycan hydrogel.

[0067] In some embodiments, the composition comprises from 10 mg / ml to 15 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml or 15 mg / ml), from 10 mg / ml to 20 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml or 20 mg / ml), from 10 mg / ml to 25 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml or 25 mg / ml), from 10 mg / ml to 30 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml or 30 mg / ml), from 10 mg / ml to 35 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml or 35 mg / ml), from 10 mg / ml to 40 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml,A biodegradable poly(α-hydroxy acid) polymer of 39 mg / ml or 40 mg / ml or from 10 mg / ml to 45 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml or 45 mg / ml). In some embodiments, the biodegradable poly(α-hydroxy acid) polymer is not gamma irradiated.,

[0068] In some embodiments, the crosslinked glycosaminoglycan hydrogel comprises crosslinked hyaluronic acid (HA), crosslinked heparan sulfate (HS), crosslinked heparin (HEP), crosslinked chondroitin sulfate (CS), crosslinked dermatan sulfate (DS), or crosslinked keratan sulfate (KS), or a combination thereof. In some embodiments, the crosslinked glycosaminoglycan hydrogel comprises crosslinked hyaluronic acid (HA).

[0069] In some embodiments, the free glycosaminoglycan comprises hyaluronic acid (HA), heparan sulfate (HS), heparin (HEP), chondroitin sulfate (CS), dermatan sulfate (DS), or keratan sulfate (KS), or a combination thereof. In some embodiments, the free glycosaminoglycan comprises hyaluronic acid (HA).

[0070] In some embodiments, the crosslinked glycosaminoglycan hydrogel comprises a crosslinked hyaluronic acid hydrogel, and the free glycosaminoglycan comprises free hyaluronic acid.

[0071] In some embodiments, the biodegradable poly(α-hydroxy acid) polymer comprises poly-L-lactic acid (PLLA), poly-D-lactic acid (PLDA), polyglycolic acid (PLGA) or polydiol citrate (PDC) or a combination thereof. In some embodiments, the biodegradable poly(α-hydroxy acid) polymer comprises poly-L-lactic acid (PLLA).

[0072] In certain embodiments, the composition comprises a hyaluronic acid (HA) gel crosslinked with 1,4 - butanediol diglycidyl ether (BDDE), free HA (i.e., uncrosslinked; about 35%), phosphate buffer, about 0.7% sodium chloride, and free poly - L - lactic acid (PLLA) (i.e., uncrosslinked).

[0073] In some embodiments, the crosslinked glycosaminoglycan hydrogel has a size of about 60 to 90 (e.g., about 60, 65, 70, 75, 80, 85, or 90) microns. In some embodiments, the term "about" means ±10% of a given value.

[0074] In some embodiments, the composition further comprises a local anesthetic at a concentration between 1 mg / ml and 15 mg / ml (e.g., 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, or 15 mg / ml, or any value therebetween), the local anesthetic being selected from lidocaine, bupivacaine, articaine, etidocaine, or carbocaine. In a specific embodiment, the composition comprises 1 mg / ml and 15 mg / ml of lidocaine.

[0075] In some embodiments, the composition further comprises sodium chloride between 0.1% and 1.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%).

[0076] In some embodiments, the composition has an osmotic pressure between about 250 mOsm / kg and 350 mOsm / kg (e.g., about 250 mOsm / kg, 275 mOsm / kg, 300 mOsm / kg, 325 mOsm / kg, or 350 mOsm / kg).

[0077] In some embodiments, the composition further comprises a phosphate buffer between 9 mM and 20 mM (e.g., 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM).

[0078] In some embodiments, the composition is a sterile composition. In some embodiments, the composition is an injectable composition. In some embodiments, the composition is packaged as a pre - filled syringe. In some embodiments, the composition can be used to treat fine lines and wrinkles among other indications.

[0079] The disclosed compositions can be used in a method for treating wrinkles or fine lines in the skin of a subject in need thereof, the treatment tightening the skin of the subject in need thereof or reducing the appearance of wrinkles or fine lines in the skin of the subject. Such a method can generally be carried out by administering the disclosed compositions to the subject by injection (e.g., subcutaneous, subcuticular, intradermal, transdermal, intramuscular, etc.). The injection can be carried out using a fine needle, such as a needle of gauge 25 to 30 (G) (e.g., 25G, 26G, 27G, 28G, 29G or 30G). The needle can be about 0.5 inches long or longer. In some embodiments, the injection can be carried out by means of a cannula.

[0080] Method for preparing a hydrogel composition

[0081] Another aspect of the disclosure relates to a method for preparing the compositions disclosed herein. In some embodiments, the method for preparing the compositions requires a higher concentration of crosslinking agent, a higher concentration of sodium chloride and a higher concentration of phosphate buffer compared to comparable filler compositions. In some embodiments, the method further comprises an additional purification / precipitation step prior to the addition of free glycosaminoglycan and free biodegradable poly(α-hydroxy acid) polymer. An exemplary flow chart illustrating an embodiment of the production method is provided in the figures.

[0082] Another aspect of the disclosure relates to a method for preparing a composition, which comprises: (a) crosslinking a glycosaminoglycan in the presence of 2% to 4% v / v (e.g., 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9% or 4%) NaOH to obtain a crosslinked glycosaminoglycan hydrogel; (b) filtering the crosslinked glycosaminoglycan hydrogel using a filter with a pore size of 60 to 90 microns; (c) adding 20% to 50% (e.g., 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%) free glycosaminoglycan by weight of the composition to the filtered crosslinked glycosaminoglycan hydrogel; and (d) adding 5 mg / ml to 60 mg / ml (5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml, 45 mg / ml, 46 mg / ml, 47 mg / ml, 48 mg / ml, 49 mg / ml, 50 mg / ml, 51 mg / ml, 52 mg / ml, 53 mg / ml, 54 mg / ml, 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml or 60 mg / ml) of a biodegradable poly(α-hydroxy acid) polymer to the composition of step (c) under vacuum conditions, wherein the biodegradable poly(α-hydroxy acid) polymer does not crosslink with the glycosaminoglycan hydrogel.

[0083] In some embodiments, the crosslinking in step (a) is achieved in the presence of 2.3% to 3.1% (e.g., 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3% or 3.1%) v / v NaOH. In some embodiments, the crosslinking in step (a) is achieved in the presence of 2.3% to 2.5% (e.g., 2.3%, 2.4% or 2.5%) v / v NaOH. In some embodiments, the crosslinking in step (a) is achieved in the presence of 2.4% NaOH.

[0084] In some embodiments, the method further comprises swelling the crosslinked glycosaminoglycan hydrogel under vacuum conditions between step (a) and step (b).

[0085] In some embodiments, the vacuum conditions are achieved by a vacuum homogenizer. In some embodiments, the vacuum conditions further include a temperature of 68°C - 72°C (e.g., 68°C, 69°C, 70°C, 71°C or 72°C).

[0086] In some embodiments, the crosslinked glycosaminoglycan and the free glycosaminoglycan are composed of the same type of glycosaminoglycan. In some embodiments, the crosslinked glycosaminoglycan is composed of a first type of glycosaminoglycan, and the free glycosaminoglycan is composed of a second type of glycosaminoglycan, wherein the first type of glycosaminoglycan is different from the second type of glycosaminoglycan.

[0087] In some embodiments, the glycosaminoglycan is gamma-irradiated. In some embodiments, the glycosaminoglycan is not gamma-irradiated.

[0088] In some embodiments, the method further comprises precipitating and washing the filtered crosslinked glycosaminoglycan hydrogel from step (b) with ethanol. In some embodiments, the washed crosslinked glycosaminoglycan hydrogel is dried and swollen (rehydrated) before step (c).

[0089] In some embodiments, the method further comprises sterilizing the composition after step (d). In some embodiments, sterilization is achieved by autoclaving the composition. In some embodiments, disinfection is achieved by ultraviolet treatment.

[0090] In some embodiments, the crosslinking in step (a) is carried out at an ambient temperature between 21°C and 25°C (e.g., 21°C, 22°C, 23°C, 24°C or 25°C).

[0091] In some embodiments, the crosslinking in step (a) is achieved between 16 hours and 30 hours (e.g., 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, or 30 hours).

[0092] In some embodiments, the composition comprises a crosslinked glycosaminoglycan hydrogel at 15 mg / ml to 17 mg / ml (e.g., 15 mg / ml, 16 mg / ml, or 17 mg / ml), 15 mg / ml to 20 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml), 15 mg / ml to 25 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, or 25 mg / ml), 15 mg / ml to 30 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, or 30 mg / ml), 15 mg / ml to 35 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, or 35 mg / ml), or 15 mg / ml to 40 mg / ml (e.g., 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, or 40 mg / ml).

[0093] In some embodiments, the glycosaminoglycan is crosslinked using a crosslinking agent. In some embodiments, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). In some embodiments, the concentration of BDDE used for crosslinking ranges from 0.001 to 0.05 g (e.g., 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, or 0.05 g) of BDDE per gram of glycosaminoglycan.

[0094] In some embodiments, the composition comprises from 20% to 30% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%), from 20% to 35% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%), from 20% to 40% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%), or from 20% to 45% (20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%) by weight of the composition of free glycosaminoglycan.

[0095] In some embodiments, the composition comprises from 10 mg / ml to 15 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, or 15 mg / ml), from 10 mg / ml to 20 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml), from 10 mg / ml to 25 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, or 25 mg / ml), from 10 mg / ml to 30 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, or 30 mg / ml), from 10 mg / ml to 35 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, or 35 mg / ml), from 10 mg / ml to 40 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml,A biodegradable poly(α-hydroxy acid) polymer of 39 mg / ml or 40 mg / ml or from 10 mg / ml to 45 mg / ml (10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml or 45 mg / ml). In some embodiments, the biodegradable poly(α-hydroxy acid) polymer is not gamma irradiated.,

[0096] In some embodiments, the crosslinked glycosaminoglycan and the free glycosaminoglycan are composed of the same type of glycosaminoglycan. In some embodiments, the crosslinked glycosaminoglycan is composed of a first type of glycosaminoglycan, and the free glycosaminoglycan is composed of a second type of glycosaminoglycan, wherein the first type of glycosaminoglycan is different from the second type of glycosaminoglycan.

[0097] In some embodiments, the crosslinked glycosaminoglycan hydrogel comprises crosslinked hyaluronic acid (HA), crosslinked heparan sulfate (HS), crosslinked heparin (HEP), crosslinked chondroitin sulfate (CS), crosslinked dermatan sulfate (DS), or crosslinked keratan sulfate (KS), or a combination thereof.

[0098] In some embodiments, the free glycosaminoglycan comprises hyaluronic acid (HA), heparan sulfate (HS), heparin (HEP), chondroitin sulfate (CS), dermatan sulfate (DS), or keratan sulfate (KS), or a combination thereof.

[0099] In some embodiments, the biodegradable poly(α-hydroxy acid) polymer comprises poly-L-lactic acid (PLLA), poly-D-lactic acid (PLDA), polyglycolic acid (PLGA) or polydiethyl citrate (PDC) or a combination thereof.

[0100] In some embodiments, the crosslinked glycosaminoglycan hydrogel has a size of about 60 to 90 (e.g., about 60, 65, 70, 75, 80, 85 or 90) microns. As used herein, the term "about" means ±10% of a given value.

[0101] In some embodiments, the biodegradable poly(α-hydroxy acid) polymer has a size of about 30 to 80 (e.g., about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80) microns. In some embodiments, the biodegradable poly(α-hydroxy acid) polymer has a size of about 40 to 63 (e.g., about 40, 45, 50, 55, 60, or 63) microns.

[0102] In some embodiments, the composition further comprises between 0.1% and 1.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%) sodium chloride.

[0103] In some embodiments, the composition has an osmotic pressure between about 250 mOsm / kg and 350 mOsm / kg (e.g., about 250 mOsm / kg, 275 mOsm / kg, 300 mOsm / kg, 325 mOsm / kg, or 350 mOsm / kg).

[0104] In some embodiments, the composition further comprises between 9 mM and 20 mM (e.g., 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM) phosphate buffer.

[0105] In some embodiments, the composition is a sterilized composition. In some embodiments, the composition is an injectable composition. In some embodiments, the composition is packaged as a pre-filled syringe. In some embodiments, the composition can be used to treat fine lines and wrinkles among other indications.

[0106] Examples

[0107] Table 1 below shows an exemplary list of some components of the disclosed composition.

[0108] Table 1 - Example Composition

[0109]

[0110] Table 2 also details the method steps used and capable of being adapted to prepare the disclosed composition. These methods are not limiting and can be adjusted as needed to account for the desired properties of the resulting composition.

[0111] Table 2 - Example Methods for Preparing the Composition

[0112]

[0113] The technology of the present invention is not limited to the specific embodiments described in this application, and these specific embodiments are intended to be separately illustrated as a single aspect of the technology of the present invention. Many modifications and variations can be made to the technology of the present invention without departing from the spirit and scope of the technology, which will be apparent to those skilled in the art. From the above description, in addition to the methods and devices enumerated herein, methods and devices that are functionally equivalent within the scope of the technology of the present invention will be apparent to those skilled in the art. Such modifications and variations are intended to fall within the scope of the technology of the present invention. It should be understood that the technology of the present invention is not limited to a specific method, reagent, compound, composition or system, and of course, the specific method, reagent, compound, composition or system can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive.

Claims

1. A composition comprising: (a) A crosslinked glycosaminoglycan hydrogel at 10 mg / ml to 45 mg / ml, (b) 20% to 50% free, non-crosslinked glycosaminoglycan by weight of the crosslinked glycosaminoglycan hydrogel, and (c) A biodegradable poly(α-hydroxy acid) polymer at 10 mg / ml to 50 mg / ml, wherein the biodegradable poly(α-hydroxy acid) polymer is not crosslinked with the glycosaminoglycan hydrogel or the free, non-crosslinked glycosaminoglycan.

2. A composition prepared by a method, the method comprising: (a) Crosslinking a glycosaminoglycan in the presence of 2% to 4% v / v NaOH to obtain a crosslinked glycosaminoglycan hydrogel; (b) Filtering the crosslinked glycosaminoglycan hydrogel using a 60-micron to 90-micron filter; (c) Adding 20% to 50% free, non-crosslinked glycosaminoglycan by weight of the crosslinked glycosaminoglycan hydrogel to the filtered crosslinked glycosaminoglycan hydrogel; and (d) Adding a biodegradable poly(α-hydroxy acid) polymer at 10 mg / ml to 50 mg / ml to the composition from (c) under vacuum conditions, wherein the biodegradable poly(α-hydroxy acid) polymer is not crosslinked with the glycosaminoglycan hydrogel.

3. The composition according to claim 2, the method further comprising swelling the crosslinked glycosaminoglycan hydrogel under vacuum conditions between (a) and (b).

4. The composition according to claim 2 or claim 3, the method further comprising precipitating and washing the filtered crosslinked glycosaminoglycan hydrogel from (b) with ethanol.

5. The composition according to any one of claims 2 to 4, further comprising sterilizing the composition after (d).

6. The composition according to any one of claims 1 to 5, wherein the composition comprises a crosslinked glycosaminoglycan hydrogel at 15 mg / ml to 20 mg / ml, 15 mg / ml to 25 mg / ml, 15 mg / ml to 30 mg / ml, 15 mg / ml to 35 mg / ml or 15 mg / ml to 40 mg / ml.

7. The composition according to any one of claims 1 to 6, wherein the composition comprises 20% to 30%, 20% to 35%, 20% to 40% or 20% to 45% free, non-crosslinked glycosaminoglycan by weight of the crosslinked glycosaminoglycan hydrogel.

8. The composition according to any one of claims 1 to 7, wherein the composition comprises a biodegradable poly(α-hydroxy acid) polymer at 10 mg / ml to 15 mg / ml, 10 mg / ml to 20 mg / ml, 10 mg / ml to 25 mg / ml, 10 mg / ml to 30 mg / ml, 10 mg / ml to 35 mg / ml, 10 mg / ml to 40 mg / mg or 10 mg / ml to 50 mg / ml.

9. The composition according to any one of claims 1 to 8, wherein the crosslinked glycosaminoglycan hydrogel comprises crosslinked hyaluronic acid (HA), crosslinked heparan sulfate (HS), crosslinked heparin (HEP), crosslinked chondroitin sulfate (CS), crosslinked dermatan sulfate (DS), or crosslinked keratan sulfate (KS), or a combination thereof.

10. The composition according to any one of claims 1 to 9, wherein the free glycosaminoglycan comprises hyaluronic acid (HA), heparan sulfate (HS), heparin (HEP), chondroitin sulfate (CS), dermatan sulfate (DS), or keratan sulfate (KS), or a combination thereof.

11. The composition according to any one of claims 1 to 10, wherein the biodegradable poly(α-hydroxy acid) polymer comprises poly-L-lactic acid (PLLA), poly-D-lactic acid (PLDA), polyglycolic acid (PLGA), or poly(diethylene citrate) (PDC), or a combination thereof.

12. The composition according to any one of claims 1 to 11, wherein the crosslinked glycosaminoglycan hydrogel has a size of about 60 to 90 microns, and optionally wherein the biodegradable poly(α-hydroxy acid) polymer is about 40 to 63 microns.

13. The composition according to any one of claims 1 to 12, wherein the crosslinked glycosaminoglycan is crosslinked by one or more multifunctional crosslinking agents.

14. The composition according to claim 13, wherein the one or more multifunctional crosslinking agents are selected from the group consisting of divinyl sulfone, polyepoxides, and diepoxides.

15. The composition according to claim 13, wherein the one or more multifunctional crosslinking agents are selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane.

16. The composition according to any one of claims 1 to 12, wherein the crosslinked glycosaminoglycan is crosslinked by (i) a non-carbohydrate-based di- or poly-nucleophilic crosslinking agent or (ii) a carbohydrate-based di- or poly-nucleophilic crosslinking agent.

17. The composition according to claim 16, wherein the non-carbohydrate-based di- or poly-nucleophilic crosslinking agent is hexamethylenediamine (HMDA).

18. The composition according to claim 16, wherein the carbohydrate-based di- or poly-nucleophilic crosslinking agent is diaminotrehalose (DATH).

19. The composition according to any one of claims 1 to 18, wherein the composition is an injectable composition.

20. A method for improving the skin quality of a subject in need thereof, the method comprising administering to the subject a composition according to any one of claims 1 to 18.

21. A method for preparing a composition, the method comprising: (a) crosslinking a glycosaminoglycan in the presence of 2% to 4% v / v NaOH to obtain a crosslinked glycosaminoglycan hydrogel; (b) Filter the cross-linked glycosaminoglycan hydrogel using a filter with a pore size of 60 to 90 microns; (c) Add 20% to 50% by weight of free glycosaminoglycan, based on the weight of the composition, to the filtered cross-linked glycosaminoglycan hydrogel; And (d) Add 10 mg / ml to 50 mg / ml of a biodegradable poly(α-hydroxy acid) polymer to the composition in step (c) under vacuum conditions, wherein the biodegradable poly(α-hydroxy acid) polymer does not cross-link with the glycosaminoglycan hydrogel.

22. The method according to claim 21, further comprising swelling the cross-linked glycosaminoglycan hydrogel under vacuum conditions between (a) and (b).

23. The method according to claim 21 or claim 22, further comprising precipitating and washing the filtered cross-linked glycosaminoglycan hydrogel from (b) with ethanol.

24. The method according to any one of claims 21 to 23, further comprising sterilizing the composition after (d).

25. The method according to any one of claims 21 to 24, wherein the composition comprises 15 mg / ml to 20 mg / ml, 15 mg / ml to 25 mg / ml, 15 mg / ml to 30 mg / ml, 15 mg / ml to 35 mg / ml, or 15 mg / ml to 40 mg / ml of cross-linked glycosaminoglycan hydrogel.

26. The method according to any one of claims 21 to 25, wherein the composition comprises 20% to 30%, 20% to 35%, 20% to 40%, or 20% to 45% by weight of free glycosaminoglycan, based on the weight of the composition.

27. The method according to any one of claims 21 to 26, wherein the composition comprises 10 mg / ml to 15 mg / ml, 10 mg / ml to 15 mg / ml, 10 mg / ml to 20 mg / ml, 10 mg / ml to 25 mg / ml, 10 mg / ml to 30 mg / ml, 10 mg / ml to 35 mg / ml, 10 mg / ml to 40 mg / ml, or 10 mg / ml to 45 mg / ml of a biodegradable poly(α-hydroxy acid) polymer.

28. The method according to any one of claims 21 to 27, wherein the cross-linked glycosaminoglycan hydrogel comprises cross-linked hyaluronic acid (HA), cross-linked heparan sulfate (HS), cross-linked heparin (HEP), cross-linked chondroitin sulfate (CS), cross-linked dermatan sulfate (DS), or cross-linked keratan sulfate (KS), or a combination thereof.

29. The method according to any one of claims 21 to 28, wherein the free glycosaminoglycan comprises hyaluronic acid (HA), heparan sulfate (HS), heparin (HEP), chondroitin sulfate (CS), dermatan sulfate (DS), or keratan sulfate (KS), or a combination thereof.

30. The method according to any one of claims 21 to 29, wherein the biodegradable poly(α-hydroxy acid) polymer comprises poly-L-lactic acid (PLLA), poly-D-lactic acid (PLDA), polyglycolic acid (PLGA), or polydiol citrate (PDC), or a combination thereof.

31. The method according to any one of claims 21 to 30, wherein the crosslinked glycosaminoglycan hydrogel has a size of about 60 to 90 microns, and optionally wherein the biodegradable poly(α-hydroxy acid) polymer is about 40 to 63 microns.

32. The method according to any one of claims 21 to 31, wherein the crosslinked glycosaminoglycan is crosslinked by one or more multifunctional crosslinking agents.

33. The method according to claim 32, wherein the one or more multifunctional crosslinking agents are selected from the group consisting of divinyl sulfone, polyepoxides, and diepoxides.

34. The method according to claim 32, wherein the one or more multifunctional crosslinking agents are selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane.

35. The method according to any one of claims 21 to 31, wherein the crosslinked glycosaminoglycan is crosslinked by (i) a non-carbohydrate-based dinucleophile or polynucleophile crosslinking agent or (ii) a carbohydrate-based dinucleophile or polynucleophile crosslinking agent.

36. The method according to claim 35, wherein the non-carbohydrate-based dinucleophile or polynucleophile crosslinking agent is hexamethylenediamine (HMDA).

37. The method according to claim 35, wherein the carbohydrate-based dinucleophile or polynucleophile crosslinking agent is diaminotrehalose (DATH).

38. The method according to any one of claims 21 to 37, further comprising formulating the composition into an injectable composition.

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